Pestiviruses, including bovine viral diarrhea virus (BVD virus, or BVDV), have been isolated from several species of animals, both domestic and wild. Identified hosts for BVDV include buffalo, antelope, reindeer and various deer species, while unique pestivirus species have been identified in giraffes and pronghorn antelope. BVDV is a small RNA virus of the family Flaviviridae. It is closely related to other pestiviruses which are the causative agents of border disease in sheep and classical swine fever in pigs. Recently a divergent pestivirus named Bungowannah pestivirus was identified as an etiologic agent of fetal infection of piglets in Australia.
Disease caused by BVDV particularly in cattle is widespread, and can be economically devastating. BVDV infection in cattle can result in breeding problems, and can cause abortions or premature births. BVDV is capable of crossing the placenta of pregnant cattle, and may result in the birth of persistently infected (PI) calves that are immunotolerant to the virus and persistently viremic for the rest of their lives. Infected cattle can also exhibit “mucosal disease”, characterized by elevated temperature, diarrhea, coughing and ulcerations of the alimentary mucosa. These persistently infected animals provide a source for dissemination of virus within the herd for further outbreaks of mucosal disease and are highly predisposed to infection with microorganisms responsible for causing enteric diseases or pneumonia.
BVDV is classified into one of two biotypes. Those of the “cp” biotype induce a cytopathic effect on cultured cells, whereas viruses of non-cytopathic, or “ncp”, biotype do not. In addition, two major genotypes (type 1 and 2) are recognized, both of which have been shown to cause a variety of clinical syndromes.
BVDV virions are 40 to 60 nm in diameter. The nucleocapsid of BVDV consists of a single molecule of RNA and the capsid protein C. The nucleocapsid is surrounded by a lipid membrane with two glycoproteins anchored in it, E1 and E2. A third glycoprotein, Ems, is loosely associated to the envelope. The genome of BVDV is approximately 12.5 kb in length, and contains a single open reading frame located between the 5′ and 3′ non-translated regions (NTRs). A polyprotein of approximately 438 kD is translated from this open reading frame, and is processed by cellular and viral proteases into at least eleven viral structural and nonstructural (NS) proteins (Tautz, et al., J. Virol. 71:5415-5422 (1997); Xu, et al, J. Virol. 71:5312-5322 (1997); Elbers, at al., J. Virol. 70:4131-4135 (1996); and Wiskerchen, et al., Virology 184:341-350 (1991)). The genomic order of BVDV is p20/Npro, p14/C, gp48/Ems, gp25/E1, gp53/E2, p54/NS2, p80/NS3, p10/NS4A, p32/NS4B, p58/NS5A and p75/NS5B. The three envelope proteins, gp48/Ems, gp25/E1 and gp53/E2, are heavily glycosylated. Ems (formerly referred to as E0 or gp48) forms homodimers, covalently linked by disulfides. The absence of a hydrophobic membrane anchor region suggests that Ems is loosely associated with the envelope. Ems induces high antibody titers in infected cattle, but the antisera has limited virus-neutralizing activity.
Among the BVDV vaccines currently available are those which contain chemically-inactivated wild-type virus. These vaccines typically require the administration of multiple doses, and result in a short-lived immune response; they also do not protect against fetal transmission of the virus. In sheep, a subunit vaccine based on a purified E2 protein has been reported. Although this vaccine appears to protect fetuses from becoming infected, protection is limited to only the homologous strain of virus, and there is no correlation between antibody titers and protection.
Modified live (ML) BVDV vaccines have been produced using virus that has been attenuated by repeated passaging in bovine or porcine cells, or by chemically-induced mutations that confer a temperature-sensitive phenotype on the virus. A single dose of a MLV BVDV vaccine has proven sufficient for providing protection from infection, and the duration of immunity can extend for years in vaccinated cattle. In addition, cross-protection has been reported using MLV vaccines (Martin, et al., In “Proceedings of the Conference of Research Workers in Animal Diseases”, 75:183 (1994)). However, existing MLV vaccines do not allow for the differentiation between vaccinated and naturally-infected animals.
Thus, it is clear that a need exists for new vaccines for controlling the spread of BVDV. Such a vaccine(s) could be invaluable in future national or regional BVDV eradication programs, and could also be combined with other cattle vaccines, representing a substantial advance in the industry. A more effective vaccine for controlling and monitoring the spread of BVDV would be a “marked” vaccine. Such a vaccine could either contain an additional antigenic determinant which is not present in wild-type virus, or lack an antigenic determinant which is present in wild-type virus. With respect to the former, vaccinated animals mount an immune response to the “marker” immunogenic determinant, while non-vaccinated animals do not. Through the use of an immunological assay directed against the marker determinant, vaccinated animals could be differentiated from non-vaccinated, naturally-infected animals by the presence of antibodies to the marker determinant. In the case of the latter strategy, animals infected with the wild-type virus mount an immune response to the marker determinant, while non-infected, vaccinated animals do not, as a result of the determinant not being present in the marked vaccine. Through the use of an immunological assay directed against the marker determinant, infected animals could be differentiated from vaccinated, non-infected animals. In both scenarios, by culling out the infected animals, the herd could, over time, become BVDV-free. In addition to the benefit of removing the threat of BVDV disease, certification of a herd as BVDV-free has direct freedom of trade economic benefits.